Conventional metrics for assessing antiproliferative drug effect have time-dependent biases that may skew results. The drug-induced proliferation rate can be used as an alternative metric for accurate and reproducible assessment of drug performance. In vitro cell proliferation assays are widely used in pharmacology, molecular biology, and drug discovery. Using theoretical modeling and experimentation, we show that current metrics of antiproliferative small molecule effect suffer from time-dependent bias, leading to inaccurate assessments of parameters such as drug potency and efficacy. We propose the drug-induced proliferation (DIP) rate, the slope of the line on a plot of cell population doublings versus time, as an alternative, time-independent metric.

Dysregulated metabolism can broadly affect therapy resistance by influencing compensatory signaling and expanding proliferation. Given many BRAF-mutated melanoma patients experience disease progression with targeted BRAF inhibitors, we hypothesized therapeutic response is related to tumor metabolic phenotype, and that altering tumor metabolism could change therapeutic outcome. We demonstrated the proliferative kinetics of BRAF-mutated melanoma cells treated with the BRAF inhibitor PLX4720 fall along a spectrum of sensitivity, providing a model system to study the interplay of metabolism and drug sensitivity. We discovered an inverse relationship between glucose availability and sensitivity to BRAF inhibition through characterization of metabolic phenotypes using nearly a dozen metabolic parameters in Principle Component Analysis. Subsequently, we generated rho0 variants that lacked functional mitochondrial respiration and increased glycolytic metabolism. The rho0 cell lines exhibited increased sensitivity to PLX4720 compared to the respiration-competent parental lines. Finally, we utilized the FDA-approved antiretroviral drug zalcitabine to suppress mitochondrial respiration and to force glycolysis in our cell line panel, resulting in increased PLX4720 sensitivity via shifts in EC50 and Hill slope metrics. Our data suggest that forcing tumor glycolysis in melanoma using zalcitabine or other similar approaches may be an adjunct to increase the efficacy of targeted BRAF therapy.

Tumor metabolism has been investigated as an exploitable avenue for treatment in the last several years. The link between tumor and metabolism traces its origin to a seminal observation by Otto Warburg, almost a century ago, that cancer cells, regardless of oxygen availability, convert most intracellular glucose to lactate—that is, aerobic glycolysis (1). This elevated glycolytic state of cancer cells has been elegantly exploited to detect tumors and their response to drugs by positron emission tomography through the use of fluorodeoxyglucose (2). Originally, tumor cells were thought to have dysfunctional mitochondria, but accumulating evidence suggests that they utilize glycolysis, oxidative phosphorylation, or both, depending on context unrelated to the integrity of mitochondria (3). Additionally, cancer cells can switch their metabolic phenotypes during tumor progression and metastasis and/or in response to external perturbations (4). This metabolic plasticity provides both the energy and the necessary intermediates for biosynthetic processes required for tumor growth. Recently, this “deregulation of cellular energetics” has been recognized as an emerging hallmark of cancer (5). Although our understanding of metabolic plasticity has increased over the years, the relationship between metabolism and gene regulatory networks (GRNs) remains understudied. In PNAS, using a systems-level approach, Jia et al. (6) explore the links between metabolism and gene regulation. Their key observation is that differential activity of the master regulators AMP-activated protein kinase (AMPK) and HIF-1 give rise to distinct metabolic phenotypes in cancer. Furthermore, based on experimentally validated model predictions, they demonstrate that cancer cells may exhibit additional metabolic states not usually present in normal cells, termed high-high or low-low. This intriguing conclusion challenges the conventional dichotomous classification of tumor metabolism as either glycolysis or oxidative phosphorylation (OXPHOS) and suggests novel avenues of experimentation. Metabolic pathways are interconnected and flexible, providing tumor cells with the property to reprogram their metabolism … [↵][1]1To whom correspondence should be addressed. Email: vito.quaranta{at}vanderbilt.edu. [1]: #xref-corresp-1-1